Effect of clomiphene citrate on human spermatozoal motility and fertilizing capacity in vitro

The effect of clomiphene citrate (CC) at various concentrations (0.005, 0.05, 0.5, 5, and 50 micrograms/ml) on the in vitro motility and fertilizing capacity of human spermatozoa was studied. Spermatozoa collected from 14 normal men were washed in modified Krebs-Ringer solution (Biggers, Whitten and Whittingham [BWW] medium) and incubated with CC for 5 hours, the period required for spermatozoal capacitation. The percent motilities of spermatozoa were recorded at 0 and 5 hours during incubation with CC. After incubation, the spermatozoa were washed with BWW medium to remove CC before insemination of the zona-free hamster ova. CC caused a significant dose-dependent decrease in the penetration of denuded hamster ova in comparison with the control (P less than 0.05). Significant depressive effect on spermatozoal motility was observed with CC at 0.05 micrograms/ml or higher concentrations (P less than 0.05). These results indicate that (1) CC decreases human spermatozoal fertilizing capacity in vitro and (2) the inhibitory effect on fertilizing capacity could be due to the sperm-immobilizing activity of CC.     

The relationship between the human sperm hypoosmotic swelling test, routine semen analysis, and the human sperm zona-free hamster ovum penetration assay

The functional integrity of sperm membranes of 270 semen samples collected from fertile men and the male partners in couples with infertile marriages was assessed by the hypoosmotic swelling test and the results correlated with routine semen analysis and the human sperm zona-free hamster ovum penetration assay. Semen samples with abnormal semen parameters had lower values of percentage of swollen sperm after hypoosmotic treatment in comparison with those with normal semen parameters. A weak positive correlation was observed between sperm swelling and sperm morphologic features (r = 0.32, P less than 0.05) and between sperm swelling and sperm motility (r = 0.22, P less than 0.05). Insignificant correlation was observed between sperm swelling and in vitro sperm fertilizing capacity, as assessed by the zona-free hamster ovum penetration assay. The results indicate that the sperm swelling test and the zona-free hamster ovum penetration assay are evaluating different functional qualities of sperm that are apparently not associated with each other.     

The application of the zona-free hamster egg test for the prognosis of human in vitro fertilization

The zona-free hamster egg test was carried out using spermatozoa from 15 men which consistently failed to fertilize their wives' oocytes in vitro. Spermatozoa from nine of these men fertilized hamster eggs in vitro, indicating that positive results in this assay are an unreliable guide to human in vitro fertilization. Donor spermatozoa were needed to fertilize the wife's oocytes in three of these cases. Nevertheless, the proportion of hamster egg penetration was significantly lower compared with spermatozoa from 15 men who could fertilize their wives' oocytes in vitro. The hamster assay also failed to indicate the establishment of pregnancy.     

A comparison between the hamster egg penetration test and the seminal parameters in men of infertile couples

The zona-free hamster egg penetration test was performed on 74 random consecutive semen samples from men of infertile couples and on 7 men of proven fertility (semen donors). Of these 74 men, 31 had never before delivered a semen sample (Group I), while the remaining 43 had delivered samples before (Group II). The penetration rates were correlated with the parameters of the semen analysis. In Group I, the mean penetration rate was 59%, in Group II, 42%, and among the fertile donors, 52%. No strong correlation was found between hamster egg penetration rates and the various semen parameters. Only the proportion of motile spermatozoa in Group II correlated significantly with the egg penetration test (P less than .05). Differences in methodology make comparison with the results from different laboratories difficult. It is likely that the egg penetration test measures some separate quality of the spermatozoa that may not be clear from the routine semen analysis. The role of the hamster egg penetration test in the investigation of the causes of infertility should be evaluated further.     

Sperm function in patients with unexplained infertility

Summary. Sperm function was studied in 27 patients with hitherto unexplained infertility. The ability of spermatozoa to reach the site of fertilization was assessed by laparoscopic sperm recovery from the peritoneal fluid and fimbrial rinsings and sperm fertilizing capacity with the zona-free hamster egg penetration in vitro test. The ability of spermatozoa to reach the site of fertilization correlated significantly with their fertilizing capacity in vitro , but was totally unrelated to any of the conventional criteria of semen quality, including the postcapacitation movement characteristics of the spermatozoa. Among patients with unexplained infertility, there are individuals with defects of sperm function which cannot be identified by conventional clinical techniques.     

In vitro penetration of human sperm into bovine cervical mucus: effects of sperm washing and exposure to low temperature

Standardized bovine cervical mucus penetration by human sperm in vitro provides information for evaluating male fertility. Normal semen specimens from 20 sperm donors and 17 infertile men were tested for cervical mucus penetration. Rigorous control of test temperature was necessary to guarantee the reliability and reproducibility of cervical mucus penetration. Sperm washing was found to significantly improve cervical mucus penetration for infertile men, from 18 +/- 2.2 to 27 +/- 3.4 mm, P less than .025. Sperm washing for normal donors had no apparent effect on cervical mucus penetration (58 +/- 1.5 mm prewash, 55 +/- 1.7 mm postwash, P greater than .1). The authors conclude that: 1) temperature for cervical mucus penetration testing is critical to reliability, and 2) cervical mucus penetration is a useful screening tool for in vitro procedures proposed to improve sperm function.     

Computer-assisted assessment of human sperm morphology: usefulness in predicting fertilizing capacity of human spermatozoa

OBJECTIVE: The usefulness of sperm morphology to predict the outcome of human sperm fertilizing capacity was examined. DESIGN, SETTING, PATIENTS: Semen samples from 50 male patients attending the infertility clinic of a tertiary referral institution were studied. MAIN OUTCOME MEASURES: Sperm morphology was classified both by visual assessment and computer-assisted image analysis. In addition, morphometric analysis of the spermatozoa was measured by the morphologizer. Multivariate discriminant analysis was used to evaluate the usefulness of these morphology parameters for predicting the outcome of the zona-free hamster oocyte sperm penetration assay. RESULTS: The manually derived percent of spermatozoa with normal and small head were selected to be of discriminating value in predicting the outcome of the zona-free hamster oocyte penetration test. The accuracy of correctly classifying the outcome of zona-free hamster oocyte penetration test by these two parameters in combination was 84%, whereas assessment of sperm morphology with morphometric analysis by the morphologizer selected a total of eight variables, which together predicted sperm fertilizing capacity with 74% accuracy. Addition of the morphologizer-derived parameters to those derived manually did not significantly improve the predictive value. CONCLUSION: We conclude that the results of the zona-free hamster egg penetration test could be predicted using manual assessment of sperm morphology and computer-assisted morphometric analysis did not add further information.     

Prolonged storage of human spermatozoa at room temperature or in a refrigerator

Spermatozoa from patients who underwent in vitro fertilization (IVF) therapy were prepared free from seminal plasma with the use of IVF culture medium supplemented with 8% human serum. Samples were then stored either at room temperature or in a refrigerator, and their motility and ability to penetrate zona-free hamster eggs were assessed every second day. With storage at room temperature, motility declined by less than half over the first 14 days, with some samples still active 20 days after preparation. The ability of the samples to penetrate hamster eggs was unchanged during the first 6 days of storage, and most of the samples still had positive tests after 14 days. Some spermatozoa could still penetrate after 17 days. Room-temperature-stored spermatozoa were still able to fertilize human oocytes 5 days after preparation. With storage in a refrigerator motility declined rapidly, and few sperm were motile after 14 days. However, these samples penetrated relatively more hamster eggs after 14 days' storage than room-temperature-stored samples did. Spermatozoa stored overnight in a refrigerator had significantly higher hamster egg penetration rates than spermatozoa stored overnight at room temperature. After storage overnight at room temperature, false-positive and false-negative results of hamster egg penetration tests were common in relation to IVF outcome; after refrigerated storage, no false-negative results were found.     

The relationship between alterations in spermatozoal deoxyribonucleic acid, heparin binding sites, and semen quality.

OBJECTIVE: To assess the relationship between spermatozoal deoxyribonucleic acid (DNA) and fertilizing potential. DESIGN: Semen samples were examined from nine fertile donors and six donors without a confirmed pregnancy. All samples were in the normal range for count, morphology, and motility. Spermatozoa from these specimens were stained with acridine orange or Feulgen's reagent. The presence of heparin binding sites was determined by counting the number of spermatozoa that bound to heparin-coated agarose beads. RESULTS: Acridine orange staining demonstrated that in the fertile group 42% +/- 2% of the spermatozoa fluoresced green indicating that the DNA was intact, whereas only 25% +/- 3% of the spermatozoa fluoresced green in the nonfertile group (P less than 0.05). Feulgen's staining revealed that more spermatozoa from infertile donors showed a heterogeneous DNA distribution (P less than 0.05). The DNA content of spermatozoa with heterogeneous distribution of DNA was reduced by 10% compared with those with homogeneous DNA (P less than 0.05). Normal spermatozoa as well as those with DNA anomalies possessed heparin binding sites. CONCLUSIONS: These data demonstrated that in donor specimens with normal counts, morphology, and motility, a higher percentage of spermatozoa possess less and/or denatured DNA in the infertile group compared with the fertile donors. In contrast, the surface membranes of spermatozoa with altered DNA have heparin binding sites as do spermatozoa with intact DNA.     

17β雌二醇对人精子功能的非基因组调节效应及其机制的研究

目的：探讨17β雌二醇对人精子功能非基因组效应的可能机制。方法：运用计算机辅助精子分析系统与人精子穿透去透明带金黄地鼠卵异种体外受精实验,评价17β雌二醇对精子功能的调节作用；精子经信号转导途径抑制剂处理后,再采用流式细胞术检测精子胞内Ca~(2+)浓度的变化。结果：1μmol/L和5μmol/L E_2-BSA能提高人精子的A级精子百分比、平均曲线运动速度、平均直线运动速度、平均路径速度(P＜0．05)；自身受精率偏低的继发不育患者精子经1μmol/L和5μmol/L E_2-BSA作用后受精率提高(P＜0．05),但对本身受精率较高的精子影响不大(P＞0．05)；应用腺苷酸环化酶、磷脂酶C和酪氨酸蛋白激酶特异性抑制剂分别作用后均明显抑制E_2-BSA引起的胞内Ca~(2+)浓度的上升(P＜0．05)。结论：17β雌二醇能显著提高精子的运动能力和受精率,17β雌二醇可能通过腺苷酸环化酶、磷脂酶C或酪氨酸蛋白激酶信号转导途径实现对人精子功能调节的非基因组效应,为改善人精子功能提供了可能的新途径。     

Cryopreservation of human spermatozoa. II. Postthaw chronology of motility and of zona-free hamster ova penetration

Postthaw dynamics of motility maintenance and ability to penetrate zona-free hamster ova were examined with human sperm. Ten semen samples were each divided into two equal volumes; one was cryopreserved while the other half remained untreated. Frozen samples were thawed, and initial evaluations for motility and hamster egg penetration were made on both untreated and frozen-thawed samples. The time difference between the initial evaluations for the two treatment groups was approximately 30 minutes as a result of the time required to freeze and thaw aliquots. Subsequent evaluations were made 6, 12, 24, and 48 hours later. Over all times both the motility and fertilizability of cryopreserved spermatozoa were significantly reduced (P less than 0.05) when compared with those of untreated sperm. The pattern of motility loss over time was similar between untreated and frozen-thawed sperm (P greater than 0.10). Conversely, differences between untreated and frozen-thawed sperm in fertilizability patterns were dramatic (P less than 0.05). This was evidenced by penetration rates for cryopreserved sperm highest at 0 hour and decreasing over time, whereas penetration by untreated spermatozoa was lowest at 0 hour, increasing to a maximum at 24 hours. These observations may be important in the development of laboratory protocols for freezing and clinical protocols for using frozen-thawed sperm.     

Two-color fluorescence staining of lectin and anti-CD46 antibody to assess acrosomal status

OBJECTIVE: To examine potential methods for distinguishing between the acrosome reaction and acrosomal loss. DESIGN: Prospective randomized study. SETTING: Department of Obstetrics and Gynecology, Osaka University Hospital, Suita, Japan. PATIENT(S): Five healthy volunteers and 34 patients with normozoospermia who were participating in an IVF program. INTERVENTION(S): Semen samples were collected from the volunteers before the hamster egg penetration assay and from the patients at the time of IVF. MAIN OUTCOME MEASURE(S): The numbers of oocytes penetrated and spermatozoa bound were determined with the hamster egg penetration assay. Acrosomal status was assessed with two-color fluorescence staining using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and MH61 (anti-CD46 monoclonal antibody) with Texas red-conjugated antimouse immunoglobulin G antiserum. RESULT(S): The MH61 monoclonal antibody inhibited the penetration of human spermatozoa into hamster oocytes but did not reduce the number of spermatozoa bound to the zona-free hamster oocytes. Two-color fluorescence staining revealed four staining patterns of the acrosomal region. The percentage of PSA-negative/CD46-positive spermatozoa increased to a greater extent than that of PSA-negative/CD46-negative spermatozoa with an increase in the incubation time. CONCLUSION(S): Two-color fluorescence staining with FITC-PSA and the anti-CD46 monoclonal antibody may be useful for distinguishing between the acrosome reaction and acrosomal loss.     

棉酚抑制仓鼠精子顶体酶类活性及其与受精能力的关系

为了预防棉酚引起生育力不可逆的问题,我们研究了棉酚对精子顶体酶类活力与其受精能力之关系.结果表明棉酚对体外仓鼠精子穿透牛宫颈粘液及受精率有明显的抑制作用.喂服棉酚6周后,它可明显地抑制仓鼠精子的受精率,延缓睾丸提取物扩散颗粒细胞的能力,提示透明质酸酶和其他精子顶体酶类可能受到抑制.此外棉酚还可显著降低精子顶体酶和芳香基硫酸酯酶活力.这些结果表明:棉酚抑制仓鼠精子顶体酶活力与其受精率的下降相一致,其抑制作用是可逆的,并与所用棉酚剂量和服药时间呈相关性.据此可得出结论,精子顶体酶活力可用来作为监护棉酚引起不育的指标.     

Cytotoxic sperm antibodies inhibit sperm penetration of zona-free hamster eggs

Zona-free hamster egg sperm penetration assay was used to study the effects of cytotoxic sperm antibodies on egg penetration by the sperm of fertile and infertile men. Twenty-nine fertile and 9 infertile men did not have significant cytotoxic sperm antibodies in their serum and seminal plasma; 7 infertile men were positive for these antibodies in serum and seminal plasma. Two others were positive in sera, and 14 were positive in seminal plasma. Sperm from 18 of 23 (78%) infertile men with sperm antibodies had poor egg penetration (less than or equal to 20%) compared with only 6 of 38 (16%) nonautoimmune men (P less than 0.0001). Sperm from nonautoimmune fertile men were coated with seminal plasma and serum of autoimmune men and serum of isoimmune women, resulting in a significant decrease in hamster egg penetration. Sixteen of 21 (76%) seminal plasma samples with cytotoxic sperm antibodies reduced the control sperm penetration of hamster eggs by greater than or equal to 50%. Coating of sperm from fertile men with serum and seminal plasma samples from non-sperm-immune fertile and infertile subjects did not alter their penetration of hamster eggs. Coating of sperm from autoimmune men with cytotoxic antibody-positive autologous seminal plasma samples resulted in a significant decrease of egg penetration. The inhibitory effect of antibody-positive seminal plasma samples on egg penetration by control sperm was abrogated when the samples were preabsorbed with sperm. It is concluded that cytotoxic sperm antibodies, especially those in seminal plasma, inhibit hamster egg penetration by autologous and control sperm. This may explain in part the incidence of infertility associated with sperm antibodies.     

人精子中芳香化酶表达与精子功能的关系

目的:研究精子细胞色素P450芳香化酶(P450arom)的表达及其与精子功能状态和受精力的关系。方法:以人精子穿透去透明带金黄地鼠卵异种体外受精试验(SPA)检测精子受精力;以三色法染色观察精子顶体反应(AR)的发生率。采用RT-PCR,用P450arom/GAPDH光密度值的比值代表P450arom的表达水平。结果:精子P450arom表达水平与受精率有一定的相关性(生育组r=0.5622;不育组r=0.6071)。正常男性与不明原因不育症患者精子P450arom/GAPDH比值分别为0.60±0.29,0.39±0.16,有显著差异(P<0.02)。P450arom表达水平与精子AR的发生率也有一定相关性(生育组r=0.5817;不育组r=0.5535)。结论:人精子中存在着P450arom表达产物;P450arom可能与精子功能有一定关系;P450arom表达异常可能与一些不明原因不育有关。     

Analysis of human spermatozoal fertilizing ability using zona-free ova

An in vitro fertilization assay employing zona-free hamster eggs was used to analyze human spermatozoal fertilizing ability. Human spermatozoa were preincubated for 18 to 20 hours in Biggers, Whitten, and Whittingham's medium (1971) at a concentration of 1 X 10(7) sperm/ml prior to the addition of zona-free superovulated hamster eggs. Eggs were examined microscopically 2 hours later for evidence of swelling or decondensing sperm heads in the cytoplasm. A total of 6266 eggs were examined in assays for both suspected fertile and infertile donors; 50 eggs/sample were examined. The percentage fertilization was found to range from 14% to 100% in the suspected fertile group with an average of 56.3%. The sperm concentration in this fertile group ranged from 22 to 303 million/ml with an average of 114. The suspected infertile samples yielded fertilization rates of 10% or less and an average count of 50.6 million/ml. These data suggest that human spermatozoa fuse with the vitelline membrane of zona-free hamster eggs and decondense with varying efficiencies. The percentage of fertilization in this cross-species system did not show a significant correlation with sperm concentration or motility. However, suspected infertile samples always yielded 10% or less fertilization in this assay. This method may have potential value as a diagnostic tool in evaluating human spermatozoal fertilizing capacity which avoids the ethical and logistcal problems associated with fertilization of human eggs in vitro.     

Role of sperm passage through cervical mucus: fertilizing capacity tested by in vitro fertilization with zona-free hamster eggs

The effect of sperm passage through cervical mucus (CM) on the fertilizing capacity of human spermatozoa was examined in the in vitro fertilization system of zona-free hamster eggs. Each drop of ejaculated semen and BWW culture medium was connected by a small capillary tube filled with preovulatory CM, egg white or BWW medium under liquid paraffin oil in a plastic petri dish. After 2 hours, zona-free hamster eggs were added to the drop of BWW culture medium containing spermatozoa which had passed through the capillary tube and the mixture was incubated for various lengths of time at 37 degrees C under 5% CO2 in air. Human spermatozoa, which were washed and preincubated for 2 hours in BWW medium, were capable of fertilizing zona-free hamster eggs but needed a longer incubation time than spermatozoa which had passed through CM. Fertilization rates of spermatozoa which had passed through CM and egg white were very similar, but no fertilization occurred in the drop containing spermatozoa which had passed through BWW medium, presumably because of the contamination with seminal plasma. These results indicate that the most important role of CM may be to separate motile spermatozoa from seminal plasma components hostile to fertilization.     

The effect of Mycoplasma hominis and Ureaplasma urealyticum on hamster egg in vitro penetration by human spermatozoa

The effects of some genital mycoplasmas on the in vitro penetration of human spermatozoa into the master egg were studied. Ureaplasma urealyticum serotypes 4, 8, and 6 showed high interfering activity: 6.3% (P less than 0.01), 12.3%, and 14.5%, respectively, against the 55.6% penetration rate of untreated sperm. Neither a cytotoxic effect of mycoplasmas on gametes nor a masking of the binding sites on the egg surface were demonstrated. In experiments carried out with U. urealyticum serotype 4, the production of diffusible relatively heat-labile factor(s) responsible for the inhibition of sperm penetration was postulated.     

Effects of the addition of serum albumin on penetration of human spermatozoa into zona-free hamster eggs

Since Yanagimachi et al. (1976) suggested that human spermatozoa were capable of penetrating into zona-free hamster eggs, this in vitro assay has been used to analyse the fertilizing ability of human spermatozoa. Serum albumin is an important constituent of the medium used for the assay. However, a great variation in the rate of sperm penetration was observed in the use of different albumin preparations at different concentrations. Therefore we examined the effects of three different kinds of albumin preparations on the rate of human sperm penetration into zona-free hamster eggs. The results obtained were as follows. The percentages of eggs being penetrated by spermatozoa from three fertile donors A,B, and C, were assessed. When Fraction V, Globulin Free and Fatty Acid Free albumin preparations were tested at a concentration of 3.5% (W/V) by the assay using sperm from donor A, penetration rates were 13.3%, 97.4%, and 8.7% respectively. Dilution of the albumin concentration to 0.3% considerably changed the penetration rates to 64.4%, 78.8% and 12.1% in that order. In cases B and C, penetration rates showed the same tendency as in case A. alpha 1 and alpha 2 globulin fractions contaminated in the Fraction V preparation possibly inhibit the human sperm penetration into zona-free hamster eggs. An appropriate quantity of fatty acid is necessary for human spermatozoa to penetrate into zona-free hamster eggs, because penetration rates were low in the percentage of Fatty Acid Free albumin regardless of the concentration added. It is concluded that use of the same preparation of good quality is mandatory for human sperm penetration tests using zona-free hamster eggs to evaluate the results with reproducibility and accuracy.     

Sperm function tests

Basing on the newer literature a survey is given concerning several methods of functional characterization of the spermatozoa. The evaluation of sperm movement characteristics, the examination of sperm-cervical mucus interaction in vitro, the zona-free hamster egg penetration test and the measurement of adenosine triphosphate levels may provide clinically valuable information. The combination of information from the conventional semen analysis with these tests of sperm function was accurate in predicting the in vivo fertilizing ability of an ejaculate. The tests are more technique consuming and expensive, therefore they are not widely available in the andrological practice.